| Objective:To synthesize the peptides conjugated chitosan and prepare nanoparticles loaded with ligustrazine(TMP)which has stable physicochemical properties(pEGF-CS-TMF-NPs).Study the mechanism of its multidrug resistance in tumor cells.Method:1.Used glutathione as the coupling agent to preparare thiolated chitosan by the reaction of amide with the optimized technology.The epidermal growth factor receptor(EGFR)-specific ligand polypeptide(pEGF)is modified by two disulfide bonds in the molecule.To analysis the structural characterization of the product by infrared spectroscopy(IR)and nuclear magnetic resonance spectroscopy.2.pEGF-CS-TMP-NPs was prepared by ionic gelation,and its morphology was observed by transmission electron microscope(TEM),The grain size,dispersion degree and the zeta potential were detected by transmission electron microscope.The entrapment efficiency and loading capacity were tested by the high performance liquid chromatography(HPLC).3.Select human breast cancer resistant cells(MCF-7/ADR)with high expression of EGFR and human leukemia drug resistant cells K562/ADR with negative expression of EGFR.MTT assay was used to determine the cell of multiple drug resistance:two resistant cells were treated with 0.10,1.00,10.00,50.00,100.00 ?mol·L-1 adriamycin for 48 h.And sensitive cells were treated with 0.05,0.10,0.50,1.00,10.00?mol-L-1 adriamycin for 48 h.Cells survival rate,IC50 of the four kinds of senstive and multiple drug resistance cells were calculated respectively.4.The cytotoxicity of pEGF-CS-TMP-NPs was detected by MTT.MCF-7/ADR and K562/ADR cells were added by 0.03,0.06,0.12,0.25,0.50 ?g·mL-1 pEGF-CS-TMP-NPs.The cells were treated for 48 h,then using the eliasa at 492 nm to detect the absorbance value.IC95,IC90,IC85 and IC80 were calculated by Origin software.5.Used the MTT method to detect the enhancement effect of pEGF-CS-TMP-NPs on the anti-tumor effect of adriamycin.Selection of MCF-7/ADR,K562/ADR,each cell was divided into four groups,The first group was treated with pEGF-CS-TMP-NPs(0.03125,0.0625,0.125,0.25 ?g·mL-1)and adriamycin(1?g·mL-1).The second group was treated with adriamycin 1?g·mL-1).The third group treated with pEGF-CS-TMP-NPs(0.0625 ?g·mL-1).The fourth group was the blank group which was only added medium and cells.for 48 h,then using the eliasa at 492 nm to detect the absorbance value.6.The reversal index of adriamycin was determined by MTT method.Selection of MCF-7/ADR,K562/ADR,each cell was divided into two groups,and were given nanoparticles non-toxic doses IC95,ICs5 and the drug were added(0,0.1,1.0,10.0,50.0,100.0 ?g·mL-1)of different concentrations of adriamycin after 24 h and continue to culture to 48 h,and detected the absorbance value by eliasa at 492 nm.The cell survival rate of each group was calculated,the standard curve was drawn by computer Origin software,and the IC50 of two kinds of cells were calculated respectively,and the reversal index of adriamycin was calculated.7.Used Western Blotting to detect the protein levels of P-gp and EGFR in MCF-7,MCF-7/ADR,K562,K562/ADR cells.8.P-gp expression levels of two kinds of drug resistant cells were detected by Western Blotting method.MCF-7/ADR and K562/ADR cells were given IC95,IC90,IC85 three concentrations of pEGF-CS-TMP-NPs.After 48 h,the protein was extracted and P-gp expression levels of the two resistant cells was detected in different concentration groupsResult:1.Synthesis of thiolated chitosan.The infrared scanning patterns showed that the-NH2 and-OH of the chitosan and the thiolated chitosan had a flexible vibration peak,while the new C-N and C=O stretching vibration peak appeared in the 1626 cm-1 and 1516 cm-1.NMR spectra showed that thiolated chitosan had three new displacement value,for the displacement of the three methylene,?2.7 is with two methylene groups adjacent to the-SH,?1.3 was imine adjacent methylene respectively,indicated that sulfhydryl groups was already connected to the surface of chitosan.When the pH value was 6.0 and reactant thiolated chitosan,glutathione,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxy acyl imines with the quality ratio of 1:1:3:1,the content of the sulfhydryl of the synthesis of thiolated chitosan thiol was the highest after the process optimization.Compared with chitosan,the synthetic peptide conjugated chitosan had three new 1H NMR peaks in the 87.0,?6.8,?6.7 The results showed that it was the aromatic hydrogen of the amino acids,indicatingthat pEGF had been successfully coupled to the molecular surface of chitosan.2.The results of the laser particle size analyzer:the particle size was 150.50 + 9.3 nm,and the degree of dispersion(PDI)was 0.059±0.007.Zeta potential was 19.30±2.02 mv.,most of the nanoparticles were spherical in shapewith uniformed dispersion under the transmission electron microscope.The results of High performance liquid chromatography(HPLC)method showed that the entrapment efficiency of chitosan nanoparticles 37.66±0.53%,and the drug loading was 3.25 ±0.34%.3.The results of MTT assay showed that the IC50 of MCF-7,MCF-7/ADR,K562,K562/ADR cells were 0.703 and 65.78,0.93,>100 ?mol·L-1 respectively.The drug resistance of MCF-7/ADR was calculated to be 93.5 times,and the drug resistance of K562/ADR was more than 100 times.4.pEGF-CS-TMP-NPs excert less cytotoxic effect on MCF-7/ADR and K562/ADR cells.With the concentration of nanoparticle for 0.06,0.12,0.25 ?g·mL-1,the sensitivity of MCF-7/ADR was higher than that of K562/ADR,which may be related with the active targeting of nanoparticles.When the concentration of nanoparticles increased to 0.50 ?g·mL-1,the sensitivity of cells to nanoparticles showed no significant difference.The effect of PEGF-CS-TMP-NPs on MCF-7/ADR cells showed that IC95,IC90,IC85 and IC80 was 0.03,0.08,0.16,0.24 ?g·mL-1 respectively.On K562/ADR cells,the IC95,IC90,IC85 and IC80 was 0.08,0.16,0.24,0.30 ?g·mL-1 respectively.5.The results showed that pEGF-CS-TMP-NPs combined with Adriamycin could significantly decrease cell viability.The effect was positively correlated to the concentration of nanoparticles with the sensitization effect of pEGF-CS-TMP-NPs to adriamycin.The sensitivity to the MCF-7/ADR cells with the positive expression of EGFR expression was significantly higher than that of K562/ADR with the negative expression of EGFR,which was related to the active targeting of the nanoparticles.6.Reversal index of MCF-7/ADR and K562/ADR was 1.21 and 1.31 with the concentration of IC95 of the pEGF-CS-TMP-NPs,and there was no significant difference between the reversal index.However,reversal index of MCF-7/ADR and K562/ADR was 3.68 and 1.87 respectivly.The sensitivity of drug resistant of pEGF-CS-TMP-NPs on MCF-7/ADR was significantly higher than that of K562/ADR cells.7.Western blotting results showed that P-gp in MCF-7/ADR and K562/ADR cells were highly expressed,while was negative expressed in MCF-7 cells and K562 cells.EGFR in MCF-7 cells and MCF-7/ADR was positively expressed,and in K562 cells and K562/ADR cells was negatively expressed.The expression level of EGFR in cell MCF-7/ADR was significantly higher than that in MCF-7 cells.8.The expression of P-gp was detected by Western Blotting after pEGF-CS-TMP-NPs was administered.The results showed that the expression level of P-gp was significantly decreased in MCF-7/ADR cells with the positive expression of EGFR,while no significant change was detected in K562/ADR cells with the negative expression of EGFR.Therefore it was concluded that pEGF-CS-TMP-NPs can reverse the tumor MDR,and the effect was related to the down-regulating tumor drug resistant protein P-gp.Conclusion:In this experiment,the low toxicity and low price of glutathione were used to prepare the chitosan,and the synthesis of the polypeptide coupled chitosan and the preparation of pEGF-CS-TMP-NPs were completed.pEGF-CS-TMP-NPs had the stable physical and chemical properties,and was dispersed evenly with a certain slow controlled release.In vitro,it was showed that pEGF-CS-TMP-NPs could reverse MDR of MCF-7/ADR cells by the peptide modified chitosan nanoparticles,Moreover the reversal effect may be related to the down-regulation of multidrug resistance associated protein P-gp expression level. |
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